CN112710843B - Plate-type immunofluorescence kit for detecting group reactivity antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a technical platform for establishing plate type immunofluorescence reaction-PCR instrument detection, and provides a plate type immunofluorescence kit for detecting group reactivity antibody detection and a preparation method thereof. According to the method and the kit for detecting the group reactive antibodies by the plate-type immunofluorescence reaction-PCR instrument, the purified HLA-I and HLA-II mixed antigen is used for quickly screening samples, the reaction of the HLA-I and HLA-II antibodies can be more accurately distinguished and detected through fluorescence values, and the kit and the method are high in sensitivity and good in specificity and repeatability.

Description

Plate-type immunofluorescence kit for detecting group reactivity antibody and preparation method thereof

Technical Field

The invention relates to the technical field of in-vitro detection, in particular to a technical platform based on plate-type immunofluorescence reaction-PCR instrument detection, and provides a plate-type immunofluorescence kit for detecting a group reactivity antibody and a preparation method thereof.

Background

After development for over fifty years, allogenic organ transplantation has become a radical treatment method for treating various organ tissue sexual failures, and the possible rejection reaction of the organ tissues directly relates to the success of clinical transplantation. When the recipient's immune system contacts alloantigens on the allograft, an anti-donor immune response is generated, and Human Leukocyte Antigen (HLA) peptides in the donor are presented to T cells and B cells, which in turn, after a series of responses, stimulate the production of HLA antibodies in the anti-donor that mediate acute and chronic rejection reactions, resulting in graft failure or reduced graft survival. The greater the HLA compatibility between the donor and recipient, the higher the success rate of transplantation.

The Population Reactive Antibody (PRA) can reflect the sensitization state of HLA antigens of a receptor, predict rejection reaction before transplantation and early warning graft loss after transplantation, effectively reduce the occurrence of various rejection reactions of organ transplantation, and is also an optimal method for preventing the occurrence of humoral rejection reaction. PRA studies began with Miescher first finding in 1954 that leukocyte agglutination antibodies were present in the serum of women who had been transfused multiple times. In 1957 Payne discovered that pregnant women also had anti-leukocyte antibodies, which were later confirmed to be anti-HLA antigens. With the gradual and intensive research, the role of PRA in humoral immunity is gradually emphasized, and PRA analysis also becomes an important standard for judging whether an immune factor exists in graft rejection.

The detection of Population Reactive Antibodies (PRA) is a current general method for evaluating the anti-HLA level at home and abroad, and the domestic main detection methods comprise an enzyme-linked immunosorbent assay and a flow cytometry method. The enzyme linked immunosorbent assay is to coat HLA antigens in a special Terasaki micropore plate, add the serum to be detected into a hole to react with the coated HLA antigens in the hole, develop the color through the action of enzyme and a substrate, and interpret the result by a matched Biotek enzyme-labeling instrument. The flow cytometry method is to coat HLA monovalent antigen on the surfaces of different microbeads, combine specific anti-HLA antibody in serum to be detected with HLA coated on the surfaces of the microbeads through reaction, perform incubation reaction with secondary antibody labeled with fluorescence, and interpret results according to fluorescence intensity by Luminex. The Terasaki microplate special for the first method is small in size, is not suitable for a general microplate reader, requires a laboratory to be matched with a Biotek microplate reader to interpret results, has reaction time close to 2 hours, and consumes a long time. The second method has the advantages of shortened reaction by half, improved specificity and sensitivity, high laboratory requirement and high price of matched instruments.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, establish a technical platform for plate-type immunofluorescence reaction-PCR instrument detection, and provide a plate-type immunofluorescence kit for detecting a group antibody and a preparation method thereof.

The technical scheme of the invention is as follows:

the invention establishes a technical platform for detecting a plate-type immunofluorescence reaction-PCR instrument, and provides a plate-type immunofluorescence kit for detecting a group reactive antibody and a preparation method thereof.

Wherein, the 96-hole is divided into a reaction hole, a positive quality control hole and a negative quality control hole.

Wherein, the reaction holes are respectively coated with the following components:

1) An expression protein of a characteristic dominant sequence of a gene encoding an HLA-class I mixed antigen; the HLA-I antigen types are shown in Table 1; the nucleotide sequence of the characteristic dominant sequence of the coding gene of the HLA-I mixed antigen is shown as SEQ ID No.1-SEQ ID No.11 in the following table 2;

2) An expression protein of a characteristic dominant sequence of a gene encoding an HLA-II mixed antigen; the HLA-II antigen types are shown in Table 1; the nucleotide sequence of the characteristic dominant sequence of the coding gene of the HLA-II mixed antigen is shown as SEQ ID No.12-SEQ ID No.17 in the following table 2;

the positive quality control hole is coated with human IgG;

the negative quality control hole is coated with albumin.

TABLE 1 HLA antigen Table

The genes encoding HLA-I and HLA-II antigens in Table 1 were retrieved from IMGT/HLA.

The concept of selecting the characteristic dominant sequences of the genes encoding HLA-I and HLA-II antigens is as follows: the antigenic determinant reaction epitope of each antigenic protein is predicted by DNAstar-protein, and the gene segment without reaction epitope is excluded. Then, the N-glycosylation sites and the O-glycosylation sites of the coding genes of the antigen proteins are predicted through NetOGlyc and NetNGlyc, and fragments containing reaction epitopes of the N-glycosylation sites and the O-glycosylation sites are excluded. Because of the high homology among HLA-I, II antigens, the fragments with high epitope homology are selected from the coding genes of the rest antigen proteins as the characteristic dominant sequences of HLA-I mixed antigens and HLA-II mixed antigens, as shown in Table 2:

TABLE 2 HLA-I and class II Mixed antigen characteristic sequences

Wherein the fluorescein-labeled antibody is a rabbit anti-human IgG polyclonal antibody.

Wherein the fluorescein is AlexaFluor-647 fluorescein. Compared with the traditional fluorescein, the fluorescein derivative has stronger brightness and signal, stronger light stability, weaker pH sensitivity and good water solubility.

The detection instrument is a fluorescence quantitative PCR instrument which is commonly equipped in the current domestic laboratories, the fluorescence detection data is stable and reliable, misreading and misreading are avoided, and missed diagnosis and misdiagnosis are reduced.

Among them, the antigen protein coated in 96-well plate is eukaryotic expression product. Although the traditional prokaryotic expression can obtain an expression product in a short time and the method is simpler, the expressed protein product is not modified and does not necessarily have natural activity. The antigen protein expressed by the eukaryotic system has antigenicity, immunogenicity and functions as those of natural protein, has better specificity and sensitivity, and is better recognized by corresponding antibodies.

The antibodies in the reagent are all rabbit-derived antibodies, and compared with the traditional murine monoclonal antibodies, the rabbit-derived antibodies have remarkable advantages, including strong specificity and high affinity. In addition, the rabbit source antibody has a unique immune system and can identify more abundant antigen epitopes.

The invention has the following technical effects: because of the large number of antigens within HLA class I and HLA class II, the number of factors to be detected is also large. If each antigen is mixed, coated and reacted after being expressed, excessive coated antigen proteins can cause mutual interference, influence the normal combination of antibodies and reactive epitopes of the antigens, and can cause false negatives of HLA-I and HLA-II. And because certain homology exists between HLA-I class and HLA-II class, cross reaction can be caused, and false positive of HLA-I class and HLA-II class can be caused. According to the invention, the characteristic dominant sequence is used for screening, so that the sample can be rapidly screened, the reaction of HLA-I and HLA-II antibodies can be more accurately distinguished through the fluorescence value, and the kit has high sensitivity and good specificity and repeatability.

Drawings

FIG. 1 is a schematic numbering of wells of a 96-well plate.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example 1 screening of Gene encoding HLA-I and HLA-II Mixed antigen

HLA-I and HLA-II antigen encoding genes were retrieved from IMGT/HLA.

The antigenic determinant reaction epitope of each antigenic protein is predicted by DNAstar-protein, and the gene segment without reaction epitope is excluded. Then, the N-glycosylation sites and the O-glycosylation sites of the coding genes of the antigen proteins are predicted through NetOGlyc and NetNGlyc, and fragments containing reaction epitopes of the N-glycosylation sites and the O-glycosylation sites are excluded. Because of the high homology among HLA-I, II antigens, fragments with high epitope homology are selected from the coding genes of the remaining antigen proteins as the characteristic dominant sequences of HLA-I mixed antigens and HLA-II mixed antigens, as shown in Table 7:

TABLE 7 sequences characteristic of HLA-I and class II mixed antigens

Example 2

The specific process of protein expression of the characteristic dominant sequence is as follows:

the designed characteristic dominant sequence is designed and synthesized by general biological systems, inc., enzyme cutting sites of BamH I and Xho I (Takara corporation) are added in the primer setting for PCR amplification reaction, and the amplification product is identified by 1.5% agarose gel electrophoresis. The purified PCR product and pcDNA3.1 were digested with BamH I and Xho I, the isolated gene of interest and plasmid pcDNA3.1 (Baiohobowei Biotechnology Co., ltd.) were recovered by gel electrophoresis and subjected to T4 DNA ligase (Takara) overnight ligation at 16 ℃ to obtain a small amount of a ligation-competent cell DH 5. Alpha. For overnight culture, and the transformant was plated on a LB solid medium (10 g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH = 7.4) plate with 100. Mu.g/ml ampicillin and incubated at 37 ℃. Randomly picking out single colony, carrying out amplification culture, extracting recombinant plasmid, carrying out double enzyme digestion identification, and carrying out sequence determination. Transfecting HEK293 cells with the recombinant plasmid, simultaneously transfecting HEK293 cells with the empty vector plasmid pcDNA3.1, 37 ℃, 5% CO ₂ Under the condition, the constant temperature incubator is shaken at 120rpm for three days. The cell culture supernatant was collected by centrifugation at 2000r/min at 4 ℃. After SDS-PAGE electrophoresis separation of the expression supernatant, western blotting identification is carried out on the expression product by taking the cell culture supernatant transfected by the empty vector as a negative control. The expression product was purified by using a Ni-NTA column (GE Co.), and the purified expression product was quantified by using the Bradford method.

The specific process of protein expression of each characteristic dominant sequence SEQ ID Nos. 1-17 is also substantially the same as the above process, and the specific operation method belongs to the existing method which should be understood by those skilled in the art, and is not detailed herein.

Example 3

Diluting protein samples expressed and purified by SEQ ID No.1-17 in a multiple ratio to 4 mu g/mL, respectively coating the protein samples into an Elisa ELISA plate, coating two reaction holes and a control hole in each protein sample, coating the protein samples at 100 mu L/hole for 18h at 4 ℃, cleaning the protein samples at 200 mu L/Kong Xiye for two times, sealing the protein samples at 120 mu L/hole for 1h at 37 ℃, pouring off the sealing liquid, and patting the protein samples to be dry. mu.L of HLA antibody positive sample corresponding to the protein (SEQ ID No.1: A1 positive sample, SEQ ID No.2: A25 positive sample, SEQ ID No.3: A68 positive sample, SEQ ID No.4: B7 positive sample, SEQ ID No.5: B18 positive sample, SEQ ID No.6: C1 positive sample, SEQ ID No.7: C4 positive sample, SEQ ID No.8: C5 positive sample, SEQ ID No.9: C7 positive sample, SEQ ID No.10: C17 positive sample, SEQ ID No.11: C18 positive sample, SEQ ID No.12: DR1 positive sample, SEQ ID No.13: DR4 positive sample, SEQ ID No.14: DQ2 positive sample, SEQ ID No.15: DQ4 positive sample, SEQ ID No.16: 1 DP positive sample, SEQ ID No.17: DP2 positive sample, sample dilution), 100. Mu.L of control sample, and room temperature dilution (30-30 rpm). Add 200. Mu.L of washing solution into each well, let stand for 1 minute, pat dry, repeat 5 times, pat dry. mu.L of goat anti-human IgG labeled with HRP (general purpose biosystems, ltd.) was added at 5000-fold dilution, and incubated at room temperature (22-28 ℃ C.), 300rpm for 30 minutes. Add 200. Mu.L of washing solution into each well, let stand for 1 minute, pat dry, repeat 5 times, pat dry. 100 mu of LTMB developing solution is added into each well, and after development is carried out for 10min at room temperature, 100 mu of 2M sulfuric acid stop solution is added to stop development. The results are shown in Table 3:

TABLE 3 reactivity of purified protein samples

	1	2	3	4	5	6	7	8	9
										A	0.968	1.176	1.027	0.932	0.933	1.017	1.084	0.899	1.092
B	0.974	1.174	1.002	0.957	0.903	1.026	1.114	0.906	1.082
										C	0.055	0.062	0.067	0.068	0.068	0.073	0.073	0.068	0.06
D	0.063	0.063	0.07	0.066	0.073	0.062	0.07	0.066	0.063
										E	0.947	1.009	0.948	0.867	0.908	1.041	1.039	0.886
F	0.944	0.94	0.951	0.9	0.921	1.053	1.095	0.862
										G	0.064	0.065	0.065	0.064	0.066	0.072	0.069	0.062
H	0.057	0.061	0.072	0.062	0.057	0.061	0.057	0.066

The results show that the protein samples expressed and purified by SEQ ID No.1-17 have stronger reaction signals with corresponding positive samples of HLA antibodies, low background values and good reactivity.

In the present embodiment, the first and second electrodes are,

a washing solution component (3M phosphate buffer (pH7.5), 0.05% Tween-20);

sample diluent components (100 mM Tris-HCl, 1500mM NaCl, 0.05% Tween-20).

Example 4

The plate-type immunofluorescence kit for determining the group reactivity comprises the following independently packaged components:

(1) The 96-hole plate is coated with antigen and antibody, and the 96-hole plate is divided into a reaction hole, a positive quality control hole and a negative quality control hole.

Referring to fig. 1, 96 Kong Bao subjects are as follows:

1A-1H, 2A-2H, 5A-5H, 6A-6H, 9A-9F and 10A-10F are reaction holes, and expression proteins of characteristic dominant sequences of the coding genes of the purified HLA-I mixed antigen prepared by the method of example 2 are coated in the reaction holes (the characteristic dominant sequences are shown as SEQ ID No.1-SEQ ID No.11, and the concentration of the expression proteins is 4 mu g/mL respectively);

3A-3H, 4A-4H, 7A-7H, 8A-8H, 11A-11F and 12A-12F are reaction holes, and expression proteins of characteristic dominant sequences of the encoding genes of the purified HLA-II mixed antigen prepared by the method of example 2 are coated in the reaction holes (the characteristic dominant sequences are shown as SEQ ID No.12-SEQ ID No.17, and the concentration of the expression proteins is 4 mug/mL respectively);

9G-12G are positive quality control holes, and the holes are coated with 4 mug/mL human IgG (Fei Peng biological Co., ltd.);

9H-12H are negative quality control wells, which are coated with 4. Mu.g/mL albumin (Sigma-Aldrich).

The coating method comprises the following steps: the 96-well plate was coated for 2h at 37 ℃. After equilibration to room temperature, 100. Mu.L of the wash solution was washed twice, and 100. Mu.L of 5% BSA blocking solution was added to each well and blocked at 37 ℃ for 0.5 hour. Drying, drying in an electronic drying oven at 27 deg.C for 18h, vacuum pumping, and sealing in aluminum foil bag filled with desiccant.

(2) A phosphate buffer reagent containing AlexaFluor fluorescein labeled antibody, which was classified as rabbit anti-human IgG polyclonal antibody (500-fold dilution, general biosystems, ltd.).

(3) Concentrated phosphate wash (3M phosphate buffer (pH7.5), 0.05% Tween-20).

(4) TBST sample dilutions (100 mM Tris-HCl, 1500mM NaCl, 0.05% Tween-20).

Example 5 results of repetitive tests using the kit of example 4

1 method of experiment

(1) Diluting a sample to be tested by 3 times of a sample diluent, respectively adding 25 mu L of diluted positive samples (HLA I:1A-1H, 2A-2H HLA II:3A-3H, 4A-4H) of the same HLA-I class and HLA-II class into a reaction hole, adding 25 mu L of sample diluent into a negative quality control hole and a positive quality control hole, and incubating for 30 minutes at room temperature (22-28 ℃) at 300 rpm;

(2) If the plate is washed manually, the liquid in the hole is sucked away, 100 mu L of washing working solution is added into each hole, the mixture is kept stand for 1 minute and then patted dry, and the patting dry is carried out after 5 times of repetition. If a full-automatic plate washing machine is used, the plate is automatically washed for 5 times, and then the plate is patted dry. Cross contamination among micropores is avoided;

(3) Adding 25 mu L of rabbit anti-human IgG polyclonal antibody marked by AlexaFluor fluorescein into the reaction hole, the positive quality control hole and the negative quality control hole, incubating for 30 minutes at room temperature (22-28 ℃) and 300 rpm;

(4) Repeating the step (2) to wash the plate;

(5) The 96-well plate is placed in a sample holder of a fluorescent quantitative PCR instrument for measurement, and a fluorescence signal value is read.

The results of example 2 in the present invention embodiment are shown in table 4:

TABLE 4 test results

	1	2	3	4	9	10	11	12
									A	478779	277516	476405	254035
B	471156	267797	437937	267447
									C	496164	275600	457117	295997
D	434218	293913	491311	272677
									E	467963	297097	470871	280477
F	461319	289946	470297	276315
									G	478862	283982	467179	289131	173558	158574	175372	177252
H	461853	269590	476405	254035	7039	7073	528	6243

The results of the above analysis were carried out on the same sample in different wells, and the analysis results are shown in table 5:

TABLE 5 analysis of repeatability results

	HLA-I	HLA-II
			Average value M	466549	278634
Standard deviation SD	17292	12305
			Coefficient of variation CV	3.71％	4.42％

From the result, the plate-type immunofluorescence kit for detecting the group reactive antibody based on the technical platform of plate-type immunofluorescence reaction-PCR instrument detection has good repeatability.

Example 6 results of compliance testing using the kit of example 4

1 method of experiment

(1) Diluting the sample to be tested by 3 times of the sample diluent, adding 25 mu L of diluted 40 positive samples and 40 negative samples (reference reagent: one Lambda LATM) into the reaction holes, adding 25 mu L of sample diluent into the negative quality control holes and the positive quality control holes, and incubating for 30 minutes at room temperature (22-28 ℃) at 300 rpm;

(2) If the plate is washed manually, the liquid in the hole is sucked, 100 mu L of washing working solution is added into each hole, the plate is kept still for 1 minute and patted dry, and the patting dry is carried out after 5 times of repetition. If a full-automatic plate washing machine is used, the plate is automatically washed for 5 times, and then the plate is patted dry. Cross contamination among micropores is avoided;

(3) Adding 25 mu L of rabbit anti-human IgG polyclonal antibody marked by AlexaFluor fluorescein into the reaction hole, the positive quality control hole and the negative quality control hole, incubating for 30 minutes at room temperature (22-28 ℃) and 300 rpm;

(4) Repeating the step (2) to wash the plate;

(5) The 96-well plate was placed in a sample holder of a fluorescence quantitative PCR instrument, and measurement was performed, and the fluorescence signal value was read, and the results are shown in Table 6:

TABLE 6 comparative results

The results show that the kit can detect 38 positive samples of 40 positive samples of the control reagent One Lambda LATM, and the positive coincidence rate is 95%. The kit detects 39 negative samples of 40 negative samples of the One Lambda LATM serving as a control reagent, and the negative coincidence rate is 97.5 percent. Wherein, the positive samples with inconsistent detection results of the negative and positive samples are 2 parts, the negative samples are 1 part, the total number is 3 parts, and the total coincidence rate is 96.25 percent.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims (3)

1. A plate-type immunofluorescence reagent kit for detecting group reactivity antibodies is characterized in that,

the detection instrument of the plate-type immunofluorescence kit is a fluorescence quantitative PCR instrument,

the plate-type immunofluorescence kit comprises the following independently packaged components:

A. a 96-hole plate coated with antigen and antibody, the 96-hole plate is divided into a reaction hole, a positive quality control hole and a negative quality control hole,

the reaction holes are respectively coated with the following components:

1) An expression protein of a characteristic dominant sequence of a gene encoding an HLA-class I mixed antigen; the HLA-I mixed antigen types are shown in a table 1; the nucleotide sequence of the characteristic dominant sequence of the coding gene of the HLA-I mixed antigen is shown as SEQ ID No.1-SEQ ID No.11 in the following table 2;

2) An expression protein of a characteristic dominant sequence of a gene encoding an HLA-II mixed antigen; the HLA-II mixed antigen types are shown in a table 1; the nucleotide sequence of the characteristic dominant sequence of the coding gene of the HLA-II mixed antigen is shown as SEQ ID No.12-SEQ ID No.17 in the following table 2;

the concept of selecting the characteristic dominant sequences of the genes encoding HLA-I and HLA-II antigens is as follows: predicting the epitope reaction epitope of each antigen protein by DNAstar-protein, and excluding the gene fragment without the epitope reaction; predicting N-glycosylation sites and O-glycosylation sites for coding genes of each antigen protein through NetOGlyc and NetNGlyc, and excluding fragments containing reaction epitopes of the N-glycosylation sites and the O-glycosylation sites; because HLA-I, II antigens have high homology, fragments with high epitope reaction epitope homology are selected from the coding genes of the rest antigen proteins as the characteristic dominant sequences of HLA-I mixed antigens and HLA-II mixed antigens, as shown in Table 2;

the positive quality control hole is coated with human IgG;

the negative quality control hole is coated with albumin;

B. labeling the antibody with fluorescein;

TABLE 1

TABLE 2 characteristic sequences of HLA-I and class II mixed antigens

2. The kit of claim 1, wherein the fluorescein-labeled antibody is a rabbit anti-human IgG polyclonal antibody.

3. The kit according to claim 2, wherein the fluorescein is AlexaFluor-647 fluorescein.

Images